Many important cellular processes are regulated by cytokines, hormones and growth factors which interact with cell-surface receptors. Signal transducer and activator of transcription (STAT) proteins play a crucial role in coordinating the response of cells to cytokine receptor stimulation by acting as cytosolic messengers and nuclear transcription factors. Upon cytokine stimulation, STATs are phosphorylated on a conserved tyrosine residue. This phosphorylation can be catalyzed by the Janus (JAK) family kinases, intrinsic cellular receptor kinases or other cellular tyrosine kinases. Activated, phosphorylated STATs then dimerize and translocate to the nucleus, where they bind to DNA or act with other DNA binding proteins in multiprotein complexes. These complexes regulate gene transcription in a cascade of intracellular signaling events that ultimately affects a wide range of biological processes, including cell growth and differentiation, the immune response, antiviral activity, and homeostasis (Grimley et al., Cytokine Growth Factor Rev., 1999, 10, 131-157; Lin et al., Oncogene, 2000, 19, 2496-2504).
The STATs were originally discovered as critical players in interferon signaling mediated by cytokine receptors lacking intrinsic tyrosine kinase domains and employing the JAK kinases. To date, seven STAT family members have been described: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 (Bromberg, J. Clin. Invest., 2002, 109, 1139-1142). STAT5A (also known as mammary gland factor, MGF) and STAT5B are two distinctly encoded proteins. STAT5A was originally identified as the prolactin-stimulated ovine gland mammary gland factor MGF (Wakao et al., Embo J., 1994, 13, 2182-2191), but was subsequently characterized as member of the STAT family when it was identified as an interleukin-2 induced STAT protein (Hou et al., Immunity, 1995, 2, 321-329). STAT5B was identified as an additional member of the STAT family that is similarly induced by interleukin-2 (Lin et al., J. Biol. Chem., 1996, 271, 10738-10744). Human STAT5A and STAT5B are both localized to chromosome 17 in the band 17q11.2 and have a very similar genomic organization (Ambrosio et al., Gene, 2002, 285, 311-318; Lin et al., J. Biol. Chem., 1996, 271, 10738-10744). Human STAT5A and STAT5B share 91% identity at the amino acid level (Lin et al., J. Biol. Chem., 1996, 271, 10738-10744).
STAT5A and STAT5B transcripts are ubiquitously expressed in human tissues, including spleen, stomach, brain, skeletal muscle, liver, kidney, lung, placenta, pancreas, heart and small intestine (Ambrosio et al., Gene, 2002, 285, 311-318). STAT5 is activated in response to a variety of cytokines, hormones and growth factors, including prolactin, various interleukins, erythropoietin and granulocyte macrophage-colony stimulating factor. STAT5 has been implicated in transducing signals that affect cell proliferation, differentiation and apoptosis, particularly in the processes of hematopoiesis and immunoregulation, reproduction and lipid metabolism (Grimley et al., Cytokine Growth Factor Rev., 1999, 10, 131-157).
While STAT5A and STAT5B share a high degree of sequence homology, each STAT5 has distinct biological functions. STAT5A-deficient mice develop normally, but mammary lobuloalveolar outgrowth during pregnancy is reduced, and female mice fail to lactate after parturition due to defects in mammary gland differentiation (Liu et al., Genes Dev., 1997, 11, 179-186). These results demonstrate that STAT5A is essential for adult mammary gland development and lactogenesis. Targeted disruption of the murine STAT5B gene leads to a striking loss of multiple, sexually differentiated responses associated with the sexually dimorphic pattern of pituitary growth hormone secretion. Male STAT5B-deficient mice exhibit body growth rates and male-specific liver gene expression levels that are decreased relative to wild-type female levels, suggesting that STAT5B is necessary for the physiological effects of male growth hormone on body growth rate and liver gene expression. Only a modest decrease in growth rate is seen in STAT5B-deficient females (Udy et al., Proc. Natl. Acad. Sci. USA, 1997, 94, 7239-7244). The phenotypes of the gene disrupted mice correlate with the patterns of expression, with STAT5A highly abundant in mouse mammary tissue during lactation and STAT5B highly abundant in muscle tissue of virgin and lactating female mice and in male mice (Liu et al., Proc. Natl. Acad. Sci. USA, 1995, 92, 8831-8835).
Disruption of both STAT5A and STAT5B results in the phenotypes associated with disruption of each individual gene and also reveals that the STAT5 proteins have redundant functions in response to growth hormone and prolactin. Mice deficient in both STAT5A and STAT5B are smaller than their wild-type littermates, and the females are infertile. Peripheral T cells from these mice are unable to proliferate in response to T cell receptor engagement and interleukin-2, suggesting that STAT5 plays a role in T cell regulation (Teglund et al., Cell, 1998, 93, 841-850).
Each STAT5 gene gives rise to both long and short isoforms. These functionally distinct isoforms, which are activated in distinct populations of cells, are generated not by RNA processing but by STAT5-cleaving protease activity, also limited to distinct populations of cells. Interleukin-3 activates full-length STAT5A and STAT5B in mature myeloid cell lines and the c-terminally truncated forms in more immature myeloid cell lines (Azam et al., Immunity, 1997, 6, 691-701). These naturally occurring truncated variants can inhibit full-length STAT5 function in cultured mammalian cells but do not affect cell growth rate (Moriggl et al., Mol. Cell. Biol., 1996, 16, 5691-5700; Wang et al., Mol. Cell. Biol., 1996, 16, 6141-6148). Additionally, an alternatively spliced form of human STAT5B exists, which uses an alternative promoter and 5′ exon within the STAT5B gene. This STAT5B transcript is found only in placenta tissue (Ambrosio et al., Gene, 2002, 285, 311-318). Alternatively spliced forms of rat STAT5A have been isolated from rat mammary gland, and are designated STAT5A1 and STAT5A2 (Kazansky et al., Mol. Endocrinol., 1995, 9, 1598-1609). A STAT5B isoform that lacks the COOH— terminal 40 amino acids has been isolated from rat liver and designated STAT5BΔ40C (Ripperger et al., J. Biol. Chem., 1995, 270, 29998-30006).
The STAT proteins are not known to contribute directly to cell cycle checkpoint regulation or DNA repair. However, they contribute to tumorigenesis through their involvement in growth factor signaling, apoptosis and angiogenesis. Additionally, because this transcription factor family participates in the immune response, defective STAT activity can compromise immune surveillance and thus promote cancer cell survival. STAT5 is commonly found constitutively activated in several cancers. To date, the most common mechanism for constitutive phosphorylation and activation of STAT proteins is excessive JAK kinase activity (Bromberg, J. Clin. Invest., 2002, 109, 1139-1142).
A role for STAT5 in the process of tumor initiation and progression is demonstrated by the link between constitutive STAT5 activity and cultured cell transformation. STAT5 activation is sufficient for transformation of hematopoietic precursor cells (Spiekermann et al., Exp. Hematol., 2002, 30, 262-271). Both STAT5A and STAT5B are constitutively phosphorylated and are transcriptionally active in K562 leukemia cells (Carlesso et al., J. Exp. Med., 1996, 183, 811-820; de Groot et al., Blood, 1999, 94, 1108-1112; Weber-Nordt et al., Blood, 1996, 88, 809-816). Additionally, increased constitutive activation of STAT5 was detected in transformed human squamous epithelial cells derived from squamous cell carcinomas of the head and neck. Targeting of STAT5B, but not STAT5A, with antisense oligonucleotides inhibited the growth of these squamous epithelial cells (Leong et al., Oncogene, 2002, 21, 2846-2853).
Abnormal STAT5 activity is indeed found associated with many cancers, particularly hematopoietic malignancies. Constitutively activated STAT5 is found in cell samples taken from patients with T-cell and B-cell acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), adult T-cell leukemia (ATL), acute myeloid leukemia (AML), chronic myelocytic leukemia (CML) and acute promyelocytic-like leukemia (APL-L) (Arnould et al., Hum. Mol. Genet., 1999, 8, 1741-1749; Carlesso et al., J. Exp. Med., 1996, 183, 811-820; Chai et al., J. Immunol., 1997, 159, 4720-4728; Gouilleux-Gruart et al., Blood, 1996, 87, 1692-1697; Spiekermann et al., Clin. Cancer Res., 2003, 9, 2140-2150; Takemoto et al., Proc. Natl. Acad. Sci. USA, 1997, 94, 13897-13902; Weber-Nordt et al., Blood, 1996, 88, 809-816). Collectively, these data demonstrate the involvement of activated STAT5 in hematopoietic cancers.
One mechanism by which constitutively activated STAT5 may promote cancer cell survival is through the inhibition of apoptosis. Introduction of a constitutively activated STAT5 protects murine T lymphoma cells against dexamethasone-induced apoptosis (Demoulin et al., J. Biol. Chem., 1999, 274, 25855-25861). Conversely, blocking of tyrosine kinase signaling using a small molecule inhibitor in cells which express BCR/ABL, a constitutively active tyrosine kinase, inhibited cell growth and induced apoptosis (Donato et al., Blood, 2001, 97, 2846-2853; Huang et al., Oncogene, 2002, 21, 8804-8816). Apoptosis was correlated with the inhibition of STAT5 activation. Viral delivery of a dominantly acting STAT5 mutant to CML primary cells, a CML cell line or prostate cancer cells induces cell death, consistent with a role of STAT5 signaling in growth and survival of cancer cells (Ahonen et al., J. Biol. Chem., 2003; Huang et al., Oncogene, 2002, 21, 8804-8816).
Inappropriate activation of STAT proteins may also allow cancer cells to survive and proliferate in the absence of cytokines and growth factors. STAT5 activation is often observed in correlation with the presence the BCR/ABL chimeric oncogene that results from a chromosomal translocation. The BCR/ABL fusion is found in both CML and ALL (Coffer et al., Oncogene, 2000, 19, 2511-2522). STAT5 activation in cells derived from CML patients is strictly dependent on BCR/ABL kinase activity and strongly correlates with its ability to confer cytokine independent growth in hematopoietic cells (Carlesso et al., J. Exp. Med., 1996, 183, 811-820; Shuai et al., Oncogene, 1996, 13, 247-254). Constitutively activated STAT5 is also found in several CML-derived cell lines expressing BCR/ABL. Furthermore, BCR/ABL is expressed in peripheral blood cells from patients with AML, and constitutively activated STAT5 was found in one of these AML patients (Chai et al., J. Immunol., 1997, 159, 4720-4728). Both the alpha and beta isoforms of STAT5A and STAT5B are found expressed in cells from AML patients and are proposed to be due to alternative mRNA splicing rather than to proteolytic cleavage (Xia et al., Cancer Res., 1998, 58, 3173-3180). Additionally, STAT5 is a major target of other leukemic fusion proteins with protein tyrosine kinase activity, including the TEL-JAK2 and TEL-ABL fusion proteins, which act to inappropriately activate STAT5 (Spiekermann et al., Exp. Hematol., 2002, 30, 262-271).
A case of acute promyelocytic-like leukemia (APL-L) exhibits a structurally abnormal STAT gene that is the result of a fusion between the retinoic acid receptor alpha (RARA) gene and the STAT5B gene. Whereas STAT5B under normal circumstances is translocated to the nucleus only upon tyrosine kinase activation, the STAT5B/RARA fusion is mislocalized in the nucleus (Arnould et al., Hum. Mol. Genet., 1999, 8, 1741-1749). The fusion protein enhances STAT3 activity, which is a characteristic shared by other APL fusion proteins (Dong and Tweardy, Blood, 2002, 99, 2637-2646).
Phosphorothioate antisense oligodeoxynucleotides, 24 nucleotides in length, complementary to the start codon of either STAT5A or STAT5B, were used to inhibit STAT5A and STAT5B expression for the purpose of investigating their role in normal hematopoiesis. Downregulation of STAT5A or STAT5B following antisense oligodeoxynucleotide treatment had no effect on the viability, clonogenecity and apoptosis of cord blood hematopoietic cells (Baskiewicz-Masiuk et al., Cell Mol. Biol. Lett., 2003, 8, 317-331).
The U.S. Pat. No. 5,534,409 discloses and claims an isolated DNA which has at least about 90% sequence identity to a nucleotide sequence encoding mammary gland growth factor (MGF), also known as STAT5. Also disclosed are oligonucleotides useful for hybridization for the purpose of isolating cDNA clones encoding MGF (Groner et al., 1996).
The U.S. Pat. No. 5,618,693 claims and discloses an isolated nucleic acid encoding a human STAT5 (hSTAT5). Generally disclosed are nucleic acids for use as hybridization probes, PCR primers and therapeutic nucleic acids, whereby hStat5 nucleic acids are used to modulate, usually reduce, cellular expression or intracellular concentration or availability of active hStat5 and whereby these therapeutic nucleic acids are typically antisense nucleic acids (McKnight et al., 1997).
Disclosed and claimed in the U.S. Pat. No. 6,160,092 is a crystal of the core protein of the STAT protein in dimeric form with an 18-mer duplex DNA that contains a binding site for the STAT dimer, as well as methods of using the structural information in drug discovery and drug development. Also disclosed are nucleic acid molecules encoding STAT5A, including RNA and DNA molecules and hybridizable nucleic acid molecules with minimum length of 12 nucleotides (Chen et al., 2000).
Disclosed in the U.S. Pat. No. 6,518,021 are nucleic acid molecules encoding a human STAT5A molecule, whereby a nucleic acid molecule is a DNA or RNA sequence or a PCR primer (Thastrup et al., 2003).
The PCT publication WO 02/46466 discloses and claims a method of inhibiting cellular proliferation mediated by a BRCA/STAT complex, comprising contacting a BRCA/STAT-containing cell with an effective amount of a BRCA/STAT complex modulating compound sufficient to modulate the amount or activity of a BRCA/STAT complex in said cell, wherein said BRCA/STAT complex modulating compound is selected from the group consisting of small molecule, polypeptide and nucleic acid. This application discloses that a BRCA/STAT complex modulating compound can be a nucleic acid, such as a DNA or RNA molecule, including antisense nucleic acids, that specifically binds to a BRCA or STAT nucleic acid (Valgeirsdottir, 2002).
The US Pre-grant publication 20030105057 claims and discloses a method wherein the amount of phosphorylated RECEPTOR/PTK-STAT pathway in a cell is altered by introducing into the cell nucleic acid molecules that encode RECEPTOR/PTK-STAT proteins, including STAT5A/B, to effect the increase or decrease of the expression and/or activation of a RECEPTOR or STAT, wherein the STAT can be STAT5A/B. This application further discloses methods whereby the amount of phosphorylated RECEPTOR/PTK-STAT protein is increased or decreased by introducing into the cell an antisense nucleic acid molecule that encodes a tyrosine kinase and/or a RECEPTOR/PTK-STAT protein (Fu et al., 2003).